KR100438415B1 - Novel antiviral peptide derived from Helicobacter pylori and use thereof - Google Patents

Abstract

The present invention relates to an antiviral peptide synthesized by substituting an amino-terminal region of ribosomal protein L1 (RPL1) produced by Helicobacter pylori bacteria with other amino acids, and its use. Specifically, SEQ ID No. 2 synthesized with an amphiphilic structure that increases the hydrophobic region by substituting the hydrophilic region of HP (2-20), a peptide having amphipathic peptide, among the amino terminal sites of the RPL1 protein with a hydrophobic amino acid. Antiviral peptides and uses thereof. The antiviral peptides and derivatives thereof of the present invention have no cytotoxicity and exhibit excellent virus-cell fusion inhibitory activity and thus can be usefully used as antiviral agents that are safe for humans.

Description

Novel antiviral peptide derived from Helicobacter pylori and use according to Helicobacter pylori

The present invention relates to an antiviral peptide synthesized by substituting an amino-terminal region of ribosomal protein L1 (RPL1) produced by Helicobacter pylori bacteria with other amino acids, and its use. Specifically, SEQ ID No. 2 synthesized with an amphiphilic structure that increases the hydrophobic region by substituting the hydrophilic region of HP (2-20), a peptide having amphipathic peptide, among the amino terminal sites of the RPL1 protein with a hydrophobic amino acid. Antiviral peptides and uses thereof.

Acquired immunodeficiency virus (HIV) infection is known to play a role in boosting HIV proliferation, as well as inducing the virus itself in the form of reactivation. Such virus groups include cytomegalovirus, herpes simplex virus, shingles virus, EBV (Epstein-Barr virus) and the like. They cause pneumonia, myelitis, multiple neuropathy, adrenitis, epididymitis, cervicitis and pancreatitis, as well as esophagitis.

When first infected with HIV, about three to six weeks later, the severe sore-like symptoms of acute HIV syndrome continue for one to two weeks and then disappear, followed by an asymptomatic "clinical incubation period" for about 10 years. But even during this long incubation period, virus proliferation and immune system destruction continue, and in the last few years, complications from immunodeficiency, namely opportunistic infections and malignancies, appear.

Treatment of AIDS includes treatment for the various complications described above (opportunistic infections, tumors, neurological disorders), anti-retroviral therapy for HIV itself, and treatment aimed at restoring a compromised immune system. There is this. Among these, antiviral therapy aims to suppress viral growth as much as possible by powerful antiviral agents at the beginning of infection with a low number of viruses and low immune system destruction, ultimately eradicating HIV completely from the body and restoring / maintaining immunity. For this purpose several antiviral agents should be coadministered to prevent the development of resistance. Along with clinical effects, plasma HIV RNA levels and the number of CD4 membrane proteins in host cells are used as treatment markers.

Most of the AIDS drugs currently used in the clinic are reverse transcriptase inhibitors and protease inhibitors, and these drugs have shown many limitations in use due to the emergence of resistant viruses as well as side effects such as severe bone marrow disorders. this will it urgent (Langtry, et al, Drugs, 37, 408-450, 1989;. Whittington, et al, Drugs, 44, 656-683, 1992;... De Clercq, Biochem Pharmacol, 47, 155-169 , 1994 ). Recently, the development of the method of gene transfer has been expanding its target. Gene therapy, as in all other experimental procedures, requires all possible precautions to rule out side effects or potentially possible adverse effects that can occur in patients treated with somatic gene therapy.

In particular, in the case of AIDS, gene therapy is the most needed disease because of the immense amount of replication of the HIV virus that causes this disease and resistance to mutations during treatment. The envelope glycoproteins gp 120 and gp 41 of the deficient virus are known to be caused by the central part of the virus invading the host cell by membrane fusion caused by binding of the host cell membrane protein CD4 (Lin, et al., Antimicrob. Agents Chemother ., 40, 133-138, 1996 ; Hart, et al., Proc. Natl. Acad. Sci. USA , 88, 2189-2193, 1991 ). Experimental AIDS gene therapy prevents HIV replication in infected cells and prevents infection into healthy cells. Gene therapy methods for this are CD4 + T cells or CD34 + for transfection or ex vivo. The cells are delivered via a rodent murine retrovirus, and then the mutated cells are injected into autologous or syngeneic / allogeneic receptors. Therefore, a substance that inhibits the membrane fusion following the binding of the envelope glycoproteins gp 120 and gp 41 of the acquired immunodeficiency virus to CD4, the membrane protein of the host cell, will provide a good clue to the development of AI treatments with a new mode of action. do. Polyanionic compounds such as polysulfate and polycarboxylates are known to inhibit the binding between the envelope glycoproteins of immunodeficiency virus gp 120 and gp 41 and the host cell's membrane protein CD4. In addition to being low and not selective for type I immunodeficiency virus, further development of this compound has not been achieved (De Clercq, Antiviral Res ., 7, 1-10, 1987 ). Recently, a synthetic peptide derived from gp 41, an envelope glycoprotein of type I immunodeficiency virus, and an analogue synthetic peptide from gp 41 have been known to have potent virus-cell fusion inhibitory activity in natural products bound to triterpenoids. Studies are actively underway (Mayaux, et al., Proc. Natl. Acad. Sci. USA , 91, 3564-3568, 1994 ; Pereira, et al., FEBS Letters , 362, 243-246, 1995 ; Ryu, et al., Biochem. Biophys. Res. Commun ., 265, 625-629, 1999 ).

The idea that peptic ulcers are predominantly caused by stress and gastric acid overproduction in the last century, but recently it has been found that these peptic ulcers are caused by Helicobacter pylori (Blaser, MJ, Trends Microbiol. , 1, 255-260, 1991 ). Attention is focused on this. Helicobacter pylori is an anaerobic microorganism belonging to gram-negative bacteria and having a very slow-growing spiral and flagella. Of the many proteins produced by Helicobacter pylori, RPL1 is composed of 230 amino acids, and the amino-terminal portion of the protein has been found to be similar in structure to cecropin. It is known. The RPL1 amino terminus of Helicobacter pylori has a perfect amphiphilic helix-like structure (Putsep, K. et al., Nature , 398, 671-672, 1999 ). The amphiphilic peptide has a structure similar to the lipid component of the cell membrane. Therefore, a mechanism of action has been reported to destroy microorganisms by binding to the cell membrane lipid components of microorganisms, or by changing and affecting the potential of the cell membranes. In addition, it has been reported that the amino terminal region of the RPL1 protein of Helicobacter pylori also has antimicrobial activity (Putsep, K., et al., Nature , 398, 671-672, 1999 ).

Accordingly, the present inventors designed and synthesized peptide derivatives with amphiphilic structures that increase hydrophobic sites by substituting a specific site sequence among the amino terminal sites of the RPL1 protein of Helicobacter pylori having amphiphile with a hydrophobic amino acid, By confirming the antiviral activity and cytotoxicity (hemolytic activity) of the peptide derivative thus synthesized, the present invention was completed by revealing that the peptide derivative is a peptide that can be used as an antiviral agent.

It is an object of the present invention to provide antiviral peptides and derivatives thereof which have no cytotoxicity and which exhibit excellent virus-cell fusion inhibitory activity which can be used as antiviral agents.

Figure 1 shows a Helicobacter pylori (Helicobacter pylori) a design of the amino-terminal HP (2-20) analogs of the peptides derived from the peptides of the ribosomal protein L1 (ribosomal protein L1) of bacteria diagram (diagram) of the present invention,

FIG. 2 shows the antiviral peptide of the present invention treated with CD4-positive HeLa cells and infected with Bexonia virus expressing gp 120 and gp 41, the envelope glycoproteins of type I immunodeficiency virus on the virus surface. It is a micrograph confirming that fusion between cells is inhibited.

A: positive control,

B: HP (2-20),

C: antiviral peptide set forth in SEQ ID NO: 2 ,

D: virus control

In order to achieve the above object, the present invention provides an antiviral peptide synthesized by replacing the amino-terminal region of the RPL1 protein produced by Helicobacter pylori bacteria with other amino acids.

The present invention also provides a use of the antiviral peptides and derivatives thereof for antiviral.

In addition, the present invention provides an antiviral pharmaceutical composition comprising the antiviral peptide and derivatives thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an antiviral peptide and derivatives thereof synthesized by substituting the amino terminal portion of the RPL1 protein produced by Helicobacter pylori with other amino acids.

The present inventors synthesized an antiviral peptide having an amphiphilic structure by increasing the hydrophobic region by substituting a hydrophobic amino acid by replacing a hydrophilic region of HP (2-20) which is an amphiphilic peptide in the amino terminal region of the Helicobacter pylori-derived RPL1 protein with hydrophobic amino acids. It was.

In order to synthesize the antiviral peptide, the present inventors used a liquid phase solidification method of Merrifield using a Fmoc amino group protective container (Merrifield, RB., J. Amer. Chem. Soc. , 85, 2149, 1963). ) The antiviral peptide described in SEQ ID NO: 2 , wherein glutamine, the 17th amino acid, and the 19th amino acid, asparagine, of the HP (2-20) peptide described in SEQ ID NO: 1 were replaced with tryptophan, respectively (see FIG. 1 ). The antiviral peptide thus prepared was isolated and purified and its purity was found to be 95% or higher. The molecular weight obtained using the Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry was calculated from the amino acid sequence and It was confirmed that antiviral peptides with the correct amino acid sequence were synthesized because of concordance.

Wherein according to the present invention consists of an amino acid sequence described in SEQ ID NO: 2 is substituted with a viral peptide of 17 amino acid of the HP (2-20) peptide described in SEQ ID NO: 1, glutamine and aspartic acid to the 19th amino acid, another hydrophobic amino acid other than tryptophan And, preferably, alanine, isoleucine, methionine, phenylalanine, proline, valine and leucine.

In addition, the antiviral peptide of the present invention can be synthesized with an amphiphilic structure in which hydrophobic sites are increased by substituting specific amino acids of HP (2-20) peptides with other amino acids, preferably arginine, which is the eighth amino acid, and the tenth amino acid. The amino acid glutamic acid and the twentieth amino acid lysine can be synthesized by substituting other hydrophobic amino acids as described above.

The present invention also provides a use of the peptides and derivatives thereof for antiviral purposes.

First, in order to confirm whether the peptide of the present invention can be used for the above purposes, the present inventors measured the antiviral activity of the peptide synthesized in the present invention. To measure antiviral activity, a Fusion Assay with CD4 positive HeLa cells and Bexonia virus (vPE 16) expressing gp 120 and gp 41, the envelope glycoproteins of type I immunodeficiency virus, was performed. It was. In the present invention, there are various methods for screening antiviral agents for immunodeficiency virus. However, in the present invention, the degree of inhibiting membrane fusion following the binding between the envelope glycoproteins gp 120 and gp 41 and the host cell membrane protein CD4 is shown. The antiviral activity was confirmed by the method of measuring. However, the use of immunodeficiency virus in general laboratories poses a significant risk, so the genes expressing the envelope glycoproteins gp 120 and gp 41 of the type I immunodeficiency virus were transfected with Bexonia virus, Membrane fusion between CD4-positive HeLa cells was measured.

As a result, the antiviral peptide described in SEQ ID NO: 2 of the present invention showed about 5 times higher cell-virus fusion inhibitory activity than the HP (2-20) peptide, which was superior to dextran used as a positive control. Activity was shown (see Table 1 , Figure 2 ).

In addition, the present inventors examined the red blood cell destruction ability of the peptide to confirm whether the peptide of the present invention is cytotoxic, HP (2-20) and the antiviral peptide described in SEQ ID NO: 2 of the present invention is cytotoxic On the other hand, melittin, a bee venom used as a positive control, showed very high cytotoxicity (see Table 2 ).

From the above results, the antiviral peptide described in SEQ ID NO: 2 of the present invention not only shows excellent cell-virus fusion inhibitory effect, but also can be usefully used as an antiviral agent that is safe for human body because it is not cytotoxic.

The present invention also provides an antiviral pharmaceutical composition comprising the above antiviral peptide and derivatives thereof as an active ingredient.

The antiviral peptide and its derivatives set forth in SEQ ID NO: 2 can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

That is, the antiviral peptides and derivatives thereof according to SEQ ID NO: 2 of the present invention can be administered in various parenteral formulations, and when formulated, fillers, extenders, binders, wetting agents, disintegrants, surfactants that are commonly used It is prepared using diluents or excipients. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the anti-viral peptide and its derivatives described in SEQ ID NO: 2 can be used in combination with a number of carriers (carrier) accepted as a drug, such as physiological saline or organic solvents, in order to increase the stability or absorption, glucose, sucrose Or carbohydrates such as dextran, ascorbic acid or antioxidants such as glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the antiviral peptide described in SEQ ID NO: 2 and its derivatives is 0.1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and may be administered 1 to 3 times a day.

The total effective amount of the antiviral peptide and its derivatives set forth in SEQ ID NO: 2 in the pharmaceutical composition of the present invention is administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. It can be administered, and multiple doses can be administered by a fractionated treatment protocol with long term administration. Since the concentration of the antiviral peptide and its derivatives as set forth in SEQ ID NO: 2 is determined in consideration of various factors such as the route of administration and the number of treatments, as well as the age and health of the patient, this should be considered. One of ordinary skill in the art will then be able to determine the appropriate effective dosage for the particular use of the antiviral peptide and its derivatives as set forth in SEQ ID NO: 2 as a pharmaceutical composition.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Synthesis and Separation of Peptides

In order to synthesize an antiviral peptide having an amphiphilic structure with an increased hydrophobic region, the present inventors have shown that the hydrophilic portion of the HP (2-20) peptide described in SEQ ID NO: 1 of the amino terminal portion of the RPL1 protein derived from Helicobacter pylori is hydrophobic amino acid. Substituted by Specifically, an antiviral peptide described in SEQ ID NO: 2 in which glutamine, the 17th amino acid, and 19th amino acid, asparagine, of the HP (2-20) peptide described in SEQ ID NO: 1 was substituted with tryptophan, respectively, was prepared. Peptide synthesis was performed using Mermofield's liquid phase solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as an amino group protecting vessel (Merrifield, RB., J. Amer. Chem. Soc. , 85, 2149, 1963) ( 1 ).

Specifically, the peptide having the carboxyl terminus of the -NH ₂ form of the peptide designed in the present invention used Rink Amide MBHA-Resin as a starting material, and the OH-type peptide of the carboxyl terminus was designated Fmoc-amino acid-Wang Resin. Used as starting material. The extension of the peptide chain by the coupling of Fmoc-amino acids was carried out by DCC (N-hydroxybenzo triazole (HOBt) -dicyclo-hexycarbodiimide) method. After coupling the Fmoc-amino acid at the amino terminal of each peptide, the Fmoc group was removed with 20% NMP (piperidine / N-methyl pyrolidone) solution, washed several times with NMP and DCM (dichoromethane) and dried with nitrogen gas. A solution of trifluoroacetic acid (TFA) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added thereto and reacted for 2 to 3 hours to remove the protecting group and the peptide from the resin. After separation, the peptide was precipitated with diethylether. The crude peptide obtained by the above method was purified by reverse phase (RP) -HPLC column (Delta Pak, C ₁₈ 300 Hz, 15, in an acetonitrile gradient containing 0.05% TFA). 19.0 mm x 30 cm, Waters). The synthetic peptide was hydrolyzed at 110 ° C. using 6 N HCl, and the residue was concentrated under reduced pressure and dissolved in 0.02 N HCl to measure amino acid composition using an amino acid analyzer (Hitachi 8500 A).

As a result of confirming the purity of the peptide prepared by the above method, the purity was more than 95%, and the molecular weight obtained using MALDI mass spectrometry (Hill, et al., Rapid Commun. Mass Spectrometry , 5, 395, 1991 ) is amino acid sequence. It was confirmed that the antiviral peptide having the correct amino acid sequence was synthesized because it is consistent with the molecular weight calculated from.

Experimental Example 1 Measurement of Antiviral Activity of Peptides

In order to measure the antiviral activity of the peptide prepared in Example 1 , the present inventors have expressed a vesicle virus expressing gp 120 and gp 41, which are envelope glycoproteins of CD4 positive HeLa cells and type I immunodeficiency virus. Fusion assay with (vPE 16) was performed.

Although there are many methods for screening antiviral agents against immunodeficiency virus, in the present invention, the degree of inhibiting membrane fusion following the binding between the envelope glycoproteins gp 120, gp 41 and the host cell membrane protein CD4 of the immunodeficiency virus The antiviral activity was confirmed by the method of measuring. However, the use of immunodeficiency virus in general laboratories poses a significant risk, so the genes expressing the envelope glycoproteins gp 120 and gp 41 of the type I immunodeficiency virus were transfected with Bexonia virus, Membrane fusion between CD4-positive HeLa cells was measured.

Samples used in the Fusion assay were prepared at a concentration of 10 μM and passed through a 0.22 μm filter. CD4-positive HeLa cells with CD4 protein on their surface were cultured in Ham's medium. As the components of the medium, 10% heat-treated newborn calf serum, 100 units / ml penicillin G, 100 µg / ml streptomycin sulfate and NaHCO ₃ (1.176 g / l) were added. . Recombinant virus vPE 16 is a Bexonia virus expressing the envelope glycoproteins gp 120 and gp 41 on the surface, the stock of vPE 16 was used to collect the supernatant of vero cells infected with vPE 16. The virus titer of the supernatant was determined using a plaque assay and the stock of virus was stored at -80 ° C. To summarize the assay, CD4 positive HeLa cells (2x10 ⁵ cells / ml) in the log phase were placed in a 12-well plate and after 3 days the cells unwashed on the plate were washed off with fresh medium. 0.2 ml of the medium containing the sample was added and incubated for 30 minutes, and 10 µl of diluted vPE 16 (7.3x10 ² PFU) was added thereto, followed by further incubation for 30 minutes. Thereafter, 0.8 ml of medium was added and after 16-20 hours, fusion formation was observed under a microscope. Fusion index and the percentage of fusion inhibition (% fusion inhibition percent) were calculated by the following equations (1) and (2) .

FI = total number of cells / number of cells-1

% Fusion Factor = (1-FI ₁ / FI ₂ ) x 100

Where FI ₁ is the fusion coefficient of the sample and FI ₂ is the fusion coefficient of the control group. The results of the fusion analysis are shown in Table 1 below.

sample density Fusion factor % Fusing Negative Control 0 0.70 ± 0.05 0 HP (2-20) 10 μM 0.59 ± 0.16 16.1 ± 10.4 SEQ ID NO: 2 10 μM 0.16 ± 0.07 76.7 ± 6.55 Positive control group 100 μg / ml 0.40 ± 0.08 57.1 ± 5.65

As a result, the antiviral peptide described in SEQ ID NO: 2 of the present invention showed better activity than dextran sulfate, which had a virus-cell fusion inhibitory activity, which was hardly found in the HP (2-20) peptide, as a positive control.

Experimental Example 2 Measurement of Cytotoxicity of Antiviral Peptides

In order to confirm whether the antiviral peptide of the present invention is cytotoxic, the inventors examined the erythrocyte destructive capacity of the antiviral peptide. Specifically, dilute human red blood cells with phosphate buffer solution (PBS, pH 7.0) to a concentration of 8% and continuously dilute the peptides at a concentration of 12.5 μM / well to 1/2 for 1 hour at 37 ° C. After the reaction was centrifuged at 1,000 g and the amount of hemoglobin contained in the supernatant was investigated by measuring the absorbance at 414 nm wavelength. To to by the addition of 1% Triton -X100 was measured for absorbance in order to examine the degree of cell destruction, cell destruction by the ability of 1% Triton X-100 to 100% compared with red blood cell destruction capacity of each anti-viral peptide equation Calculated according to 3 .

In the above formula, absorbance A is the absorbance of the peptide solution at 414 nm wavelength, absorbance B is the absorbance of PBS at 414 nm wavelength and absorbance C is the absorbance of 1% Triton X-100 at 414 nm wavelength.

The results of erythrocyte destructive ability according to the above formula are shown in Table 2 below.

Cytotoxicity of Antiviral Peptides Peptide % Erythrocyte destructive capacity (μM) 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.097 HP (2-20) 0 0 0 0 0 0 0 0 Antiviral peptide of SEQ ID NO: 2 0 0 0 0 0 0 0 0 Melittin 100 100 95 93 31 0 0 0

As a result, the antiviral peptide described in HP (2-20) and SEQ ID NO: 2 of the present invention showed little cytotoxicity, whereas melittin, a bee venom used as a positive control, was highly cytotoxic. .

Experimental Example 3 Acute Toxicity in Parenteral Administered Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended parenterally in a dose of 1 g / kg / 15 ml by suspending the antiviral peptide set forth in SEQ ID NO: 2 of the invention in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hemologic and hematological examinations were performed. The necropsy was performed to visually observe the abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the antiviral peptide of the present invention showed no toxicity change to rats up to 5 mg / kg and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 10 mg / kg or more.

From the above results, the antiviral peptide described in SEQ ID NO: 2 of the present invention showed strong virus-cell fusion inhibitory activity and did not show cytotoxicity, and thus can be usefully used as a safe antiviral agent for human body.

<110> Chosun University <120> Novel antiviral peptide derived from Helicobacter pylori and use according <130> 1p-08-19B <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> PRT <213> Helicobacter pylori <220> <221> PEPTIDE <222> (1) .. (19) <223> 2-20 amino acid sequence of RPL1 protein from amino-terminal end <400> 1 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Gln 1 5 10 15 Asn Asp Lys <210> 2 <211> 19 <212> PRT <213> Helicobacter pylori <400> 2 Ala Lys Lys Val Phe Lys Arg Leu Glu Lys Leu Phe Ser Lys Ile Trp 1 5 10 15 Asn Trp Lys

Claims (8)

delete delete delete delete delete delete delete Pharmaceutical composition for anti-viral comprising the peptide described in SEQ ID NO: 2 as an active ingredient.